How to grow patient-derived organoids (PDOs)

Patient-Derived Organoids (PDOs or just “organoids”) are frequently mentioned in relation to drug discovery and medical research. This article explains what PDOs are, and how scientists derive and grow them from donated patient tissue. These 3D biology methods are relatively new, and this field of research is rapidly developing.

Learn all about how to grow PDOs including:

Organoids are 3D, microscopic versions of the organs from which they originate. For example, miniature “guts” can be derived from healthy or diseased biopsy tissue from a patient’s intestines. Importantly, PDOs are faithful replicates of the tissue they originate from, including the different cell types and any underlying disease.

The picture is of a colorectal cancer organoid or “mini-gut.”The blue stain shows the DNA in individual cells and the yellow color shows up spaces within the structure that are equivalent to the intestinal cavity. A whole 3D PDO is shown on the left and on the right you can see a slice through the middle

PDO Mini-gut organoid slice

To derive and grow organoids in the lab is laborious, technically demanding and costly. It requires the skill of experienced scientists and expensive equipment, quite apart from the ethical considerations and permissions to work with human tissue. (The Human Tissue Act 2004 regulates activities concerning the removal, storage, use and disposal of human tissue.) However, the effort is worth it, as the resulting structures are increasingly being used to help scientists to understand how the body works and as a model on which to test potential new drugs.

What are patient-derived organoids?

PDOs are 3D cell structures that arise from “adult stem cells” within patient tissue that can regenerate to maintain and repair damaged tissue. They have “pre-programmed instructions” to divide and form the specialized cell types found in their specific organ of origin. The cells, once formed, self-assemble to replicate the same “architecture” as found in the body. The structures that form are therefore called organoids because they are “like an organ.”PDOs can be derived from many organs such as the gut, heart, liver, kidney, pancreas, lung, brain and many more. The small size and faithful reproduction of patient tissue outside of the patient, allows scientists to use PDOs for multiple experiments in the lab that could not ethically be performed on the patients themselves! For example, to test and accurately predict patient responses to novel drugs.

Like a bonsai tree or a baby watermelon, PDOs are just smaller versions of the original

Like a bonsai tree or a baby watermelon, PDOs are just smaller versions of the original. A microscope is needed to be able to look at them in any detail. The diameter of a section of the small intestine of the gut is around 3 centimeters (0.03 meters) and a mini-gut organoid grown for a few days in the lab is around 75 micrometers (0.000075 meters) i.e., 400 times smaller.

Where do PDOs come from?

Tissue from which to derive organoids, is generously donated with patient consent. This is usually from a needle biopsy taken by a clinician to ascertain if the tissue is healthy or if the patient has cancer or another disease. A few millimeters of the biopsy is all that is required. The chances of establishing organoids in culture are greater with more tissue, but it still can’t be guaranteed that organoids can be derived from every sample.

Creating "mini-gut" organoids from patient intestinal tissue: A step-by-step process

The procedure for deriving organoids from patient tissue varies depending on the type of tissue. Different labs have their own preferred methods that are tailored to their requirements. The more we learn about the process, the more efficient and successful the methods are becoming. The example below is a general overview of the steps involved in creating “mini-guts” from intestinal tissue. The process, from receiving the sample in the research lab, to having a culture in the incubator, in preparation for the organoids to form, takes around 4 hours. Growth of the organoids takes around 2 weeks from tissue preparations.

Enzymatic digestion to small fragments/cells

  1. The patient biopsy tissue is allocated for the various tests needed for diagnosis. The tiny piece for derivation of organoids is placed in a special storage solution and chilled to keep the cells alive and functioning normally whilst it is transported to the research lab. (The same solution is used for whole organs needed for transplant.)
  2. In the lab, the anonymised sample is sprayed with 70% alcohol and placed in a Biological Safety Cabinet (BSC). The cabinet produces air flows that pass between the operator and the sample. This maintains isolation of both from each other.
  3. The sample is rinsed with liquid containing nutrients and salts (“media”) and chopped very finely in a Petri dish using scalpels.
  4. An enzyme solution is added, and the mixture is incubated in a water bath at body temperature (37°C) for about 30 minutes. This breaks down the structure of the tissue and releases the “adult stem cells” that will enable the organoids to form.
  5. The enzymatic digestion is stopped with media and the mixture is spun at high speed inside a centrifuge. The resulting pellet is resuspended in fresh media. This step may be repeated several times to “wash” the sample.
  6. Sample is spun rapidly to pellet the tissue fragments
  7. The pellet containing tiny clumps of cells/tissue fragments is chilled to 4°C and carefully resuspended in a gelatinous protein mixture such as Matrigel or its equivalent. Matrigel is liquid at 4°C and forms a gel at room temperature. This hydrogel provides growth factors and structural support for the cells to develop in three dimensions. The cells form into a ball that can be quite rounded like a football with or without protrusions. Some organoids can be quite irregular in their overall shape.
  8. Tiny blobs of the cell fragment and Matrigel mixture are placed onto sterile petri dishes. These blobs are approximately the size of an average drop of water i.e., 0.05mL. The blobs are allowed to gel at room temperature. A liquid feed is added. This contains nutrients and additional factors that encourage growth and development of the organoids without altering the biology of the cells. The dish is then moved to an incubator that maintains a constant temperature of 37°C and 5% Carbon Dioxide. (Standard cell culture conditions).
  9. The liquid feed is renewed every few days, and organoids will develop within about 2 weeks. The yield is very variable with anything from 0 to 100 organoids per biopsy. This can depend on the tissue type and how much there is of the original biopsy sample.

How to expand organoid numbers

Once the PDOs are fully established, they can be split apart to seed many more copies. This can be done in as little as a few days or every few weeks and varies with the organ type and with the person since PDOs derived from each patient are unique. The adult stem cells within the organoid fragments will generate new cells and form organoids in the same way as they formed from the original tissue sample, except they form more quickly after they have already been established. The process of splitting and reseeding more copies is known as “sub-culture” or “passage” and can be repeated multiple times, resulting in many additional copies. These can be used for small-scale research into genetics and disease.

Method for an organoid subculture (passaging)

  1. Dissolve the Matrigel surrounding the organoids
  2. Pass the organoids up and down through the narrow opening of a pipette tip to dissociate them mechanically or break the cell clusters apart using a chemical solution containing enzymes.
  3. Spin to pellet the cells, remove the liquid.
  4. Mix the organoid cells/fragments with fresh Matrigel and culture as before.

Expand organoids at scale using our patent-pending bioprocess technology

人工培养的类器官可能会给研究人员带来重大挑战,从而延迟实验并增加成本。最常见的挑战就是无法获得质量和大小均同质的足够数量的类器官。如果不在规范的工业环境中采用标准化、可重复的工艺,几乎就不可能实现批间一致性。

Molecular Devices 领导

MOLECULAR DEVICES 在人源性 3D 类器官的扩展和工业制造方面拥有全球领导地位。凭借我们的独特生物工艺,我们的半自动化程序使用受控制和受监控的条件来生产大量规定尺寸的标准化患者来源类器官 (PDO)。严格的质量控制可确保减少批间和用户间差异。

Learn more about our 3D Ready Organoid Expansion Service.

Discover how organoids can be used downstream in high-throughput assays

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